Nitric Oxide Regulates Cell Sensitivity to Cisplatin-Induced Apoptosis Through S-Nitrosylation and Inhibition of Bcl-2 Ubiquitination

Research Article

Nitric Oxide Regulates Cell Sensitivity to Cisplatin-Induced Apoptosis through S-Nitrosylation and Inhibition of Bcl-2 Ubiquitination

Pithi Chanvorachote,1,4 Ubonthip Nimmannit,4 Christian Stehlik,2 Liying Wang,3 Bing-Hua Jiang,2 Boonsri Ongpipatanakul,4 and Yon Rojanasakul1

1Department of Pharmaceutical Sciences and 2Mary Babb Randolph Cancer Center, West Virginia University; 3Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia; and 4Pharmaceutical Technology (International) Program, Chulalongkorn University, Bangkok, Thailand

Abstract pathways, including ATR, p53, p73, and mitogen-activated protein Cisplatin is a potent cytotoxic agent commonly used for the kinase, and culminate in the activation of cell apoptosis (3). treatment of solid tumors. However, tumor cell–acquired Cisplatin-induced cell death depends further on the generation of resistance to cisplatin-induced apoptosis is a major limitation reactive oxygen species (ROS; refs. 4, 5). However, cisplatin- for efficient therapy, as frequently observed in human lung mediated cell death is frequently impaired and is a major limitation cancer. Nitric oxide (NO) is a key regulator of apoptosis, but of cisplatin-based chemotherapy (1, 6). its role in cisplatin-induced cell death and the underlying One frequent mechanism of apoptosis resistance of tumor cells mechanism are largely unknown. Previous studies indicate is the deregulated expression of proapoptotic and antiapoptotic increased NO synthase activity and elevated NO production in proteins. Amplification and overexpression of the B-cell lympho- lung carcinomas, which correlate with the incidence of ma-2 (Bcl-2) proto-oncogene occurs in many malignancies, chemotherapeutic resistance. Here, we show that NO impairs including small cell lung carcinomas, and impairs the intrinsic the apoptotic function of cells and increases their resistance apoptotic signaling by neutralizing proapoptotic Bcl-2 family to cisplatin-induced cell death in human lung carcinoma members, such as Bax (7, 8). Bcl-2 plays a crucial role in H-460 cells. The NO donors sodium nitroprusside and cisplatin-induced apoptosis and is a key determining factor of dipropylenetriamine NONOate were able to inhibit cisplatin- cisplatin resistance (9–12). The expression of Bcl-2 is regulated by induced cell death, whereas the NO inhibitors aminoguanidine several mechanisms, including transcription, posttranslational and 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline- modifications, dimerization, and degradation. Increasing evidence 1-oxyl-3-oxide had opposite effect. Cisplatin resistance in suggest that Bcl-2 expression is mainly regulated at the posttran- H-460 cells is mediated by Bcl-2, and NO up-regulates its scriptional level by protein stability. Numerous stimuli can induce a a expression by preventing the degradation of Bcl-2 via ubiquitin- the degradation of Bcl-2, including tumor necrosis factor- (TNF- ), h proteasome pathway. Cisplatin-induced generation of reactive ROS, lipopolysaccharide, -amyloid, and ischemia (9, 13–17). a oxygen species causes dephosphorylation and degradation Degradation of Bcl-2 in response to certain stimuli, such as TNF- of Bcl-2. In contrast, generation of NO has no effect on Bcl-2 and ROS, require its dephosphorylation (16, 17). Degradation of phosphorylation but induces S-nitrosylation of the protein, Bcl-2 is mainly mediated by the ubiquitin-dependent proteasome which inhibits its ubiquitination and subsequent proteasomal complex upon the covalent attachment of ubiquitin (18, 19). degradation. These findings indicate a novel pathway for Nitric oxide (NO) is a key cellular mediator synthesized from NO regulation of Bcl-2, which provides a key mechanism for L-arginine in a reaction catalyzed by NO synthases (NOS; refs. cisplatin resistance and its potential modulation for improved 20, 21). NO has been shown to possess both proapoptotic and cancer chemotherapy. (Cancer Res 2006; 66(12): 6353-60) antiapoptotic functions, depending on the cell type and cellular redox state, as well as on the concentration and flux of NO (22, 23). Introduction Induction of apoptosis by NO was attributed to its ability to induce oxidative stress and caspase activation (24). In contrast, endoge- The platinum coordination complex cis-diamminedichloroplati- nous NO production or the exposure to appropriate amounts of NO num (cisplatin) is a widely prescribed chemotherapeutic agent. It is reportedly inhibits apoptosis, which has been shown in various generally used alone or in combination with other therapeutic in vivo and in vitro experimental models (25, 26). NOS play a role in agents for the treatment of solid tumors, such as testicular, ovarian, the production of NO in lung neoplasia and may thus influence NO- bladder, cervical head and neck, and small cell and non–small cell mediated functions in tumor tissues (27). Elevation of NOS activity lung cancer (1, 2). The cytotoxic mode of action of cisplatin is still has been reported in human lung adenocarcinomas, and increased unclear, although it was suggested that the interaction of this tumor-associated NO production has been observed in lung cancer compound with DNA forms DNA adducts, primarily intra-strand patients (25, 28, 29). Together with the high incidence of cisplatin cross-link adducts, which activate several signal transduction resistance in lung cancer, NO may play a role in regulating lung carcinoma cell sensitivity to cisplatin-mediated cell death. The mechanisms by which NO regulates apoptosis resistance to Requests for reprints: Yon Rojanasakul, West Virginia University, Morgantown, WV 26506. E-mail: [email protected] or Ubonthip Nimmannit, Chulalongkorn cisplatin have not been investigated. In the present study, we University, Bangkok 10330, Thailand. Phone: 304-293-1476; E-mail: Ubonthip.N@ determined the role of NO in cisplatin-induced apoptosis and Chula.ac.th. I2006 American Association for Cancer Research. elucidated its regulatory mechanisms using pharmacologic and doi:10.1158/0008-5472.CAN-05-4533 gene manipulation approaches. Our findings show an important www.aacrjournals.org 6353 Cancer Res 2006; 66: (12). June 15, 2006

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Cancer Research role of NO in Bcl-2 regulation and its antiapoptotic function in reader. The relative percentage of cell survival was calculated by dividing cisplatin-induced cell death. The mechanism by which NO the absorbance of treated cells by that of the control in each experiment. regulates Bcl-2 involves S-nitrosylation and inhibition of ubiq- NO and ROS detection. Intracellular NO and ROS production was uitin/proteasome–mediated degradation. Such regulation is inde- determined by flow cytometry using the NO-specific probe DAF-DA and the oxidative probe DCF-DA (Molecular Probes) according to the manufac- pendent of the dephosphorylation process, thus revealing the turer’s instructions. Briefly, cells were incubated with the probe (10 Amol/L) existence of a novel mechanism of cell death regulation, which for 30 minutes at 37jC, after which they were washed, trypsinized, might be exploited in cancer chemotherapy. resuspended in PBS (1 Â 106/mL), and immediately analyzed by flow cytometry using a 488-nm excitation beam and a 538-nm band-pass filter (FACSort, Becton Dickinson, Rutherford, NJ) with CellQuest software Materials and Methods (Becton Dickinson). Cells and reagents. NCI-H460 cells were obtained from the American Western blotting. After specific treatments, cells were incubated in lysis Type Culture Collection (Rockville, MD). Cells were cultured in RPMI 1640 buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1% Triton X-100, 150 mmol/L containing 5% fetal bovine serum, 2 mmol/L L-glutamine, and 100 units/mL NaCl, 10% glycerol, 1 mmol/L Na3VO4, 50 mmol/L NaF, 100 mmol/L penicillin/streptomycin in a 5% CO2 environment at 37jC. Sodium phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture nitroprusside, aminoguanidine, 2-(4-carboxy-phenyl)-4,4,5,5 tetramethylimi- (Roche Molecular Biochemicals) for 20 minutes on ice. After insoluble dazoline-1-oxy-3-oxide (PTIO), N-acetylcysteine (NAC), DTT, benzyloxycar- debris was pelleted by centrifugation at 14,000 Â g for 15 minutes at 4jC, bonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD-fmk), and cisplatin the supernatants were collected and determined for protein content using were obtained from Sigma Chemical, Inc. (St. Louis, MO). Dipropylenetri- the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins (40 Ag) amine NONOate was from Alexis Biochemical (San Diego, CA). Diamino- were resolved under denaturing conditions by SDS-PAGE (10%) and fluorescein diacetate (DAF-DA), dichlorofluorescein diacetate (DCF-DA), transferred onto nitrocellulose membranes (Bio-Rad). The transferred and Hoechst 33342 were obtained from Molecular Probes, Inc. (Eugene, OR). membranes were blocked for 1 hour in 5% nonfat dry milk in TBST Antibodies for Bcl-2, phospho-Bcl-2 (Ser87), myc, and peroxidase-labeled [25 mmol/L Tris-HCl (pH 7.4), 125 mmol/L NaCl, 0.05% Tween 20] and secondary antibodies and protein A-agarose were obtained from Santa Cruz incubated with the appropriate primary antibodies at 4jC overnight. Biotechnology (Santa Cruz, CA). Antibodies for ubiquitin, S-nitrosocysteine, Membranes were washed thrice with TBST for 10 minutes and incubated and h-actin were from Sigma (St. Louis, MO). The transfecting agent with horseradish peroxidase–coupled isotype-specific secondary antibodies LipofectAMINE was obtained from Invitrogen (Carlsbad, CA). for 1 hour at room temperature. The immune complexes were detected by Plasmid and transfection. The Bcl-2 plasmid was generously provided enhanced chemiluminescence detection system (Amersham Biosciences, by Dr. C. Stehlik (West Virginia University Cancer Center, Morgantown, Piscataway, NJ) and quantified using analyst/PC densitometry software WV). The open reading frame of Bcl-2 and ubiquitin were amplified by high- (Bio-Rad). Mean densitometry data from independent experiments were fidelity PCR (Stratagene, La Jolla, CA) from corresponding expressed normalized to result in cells in the control. The data were presented as the sequence tags and cloned into pcDNA3 expression vectors containing the mean F SD and analyzed by the Student’s t test.

NH2-terminal myc epitope tag. Authenticity of all constructs was verified by Immunoprecipitation. Cells are washed after treatments and lysed in DNA sequencing. Transient transfection was done using LipofectAMINE lysis buffer at 4jC for 20 minutes. After centrifugation at 14,000 Â g for 15 reagent (Invitrogen), according to the manufacturer’s instructions. The minutes at 4jC, the supernatants were collected and determined for protein amount of DNA was normalized in all transfection experiments with content. Cleared lysates were normalized, and 60 Ag proteins were pcDNA3. Expression of proteins was verified by Western blotting or incubated with 12 AL of anti-myc agarose bead (Santa Cruz Biotechnology) immunoprecipitation. diluted with 12 AL Sepharose for 4 hours at 4jC. The immune complexes Generation of stable Bcl-2 transfectant. Stable transfectant of Bcl-2 were washed with 20 volumes of lysis buffer, resuspended in 2Â Laemmli was generated by culturing H-460 cells in a six-well plate until they reached sample buffer, and boiled at 95jC for 5 minutes. Immune complexes were 80% to 90% confluence. One microgram of cytomegalovirus-neo vector and separated by 10% SDS-PAGE and analyzed by Western blot as described. 15 AL of LipofectAMINE reagent with 2 Ag of myc-tagged Bcl-2 plasmid were used to transfect the cells in each well in the absence of serum. After 10 hours, the medium was replaced with culture medium containing 5% Results fetal bovine serum. Approximately 36 hours after the beginning of the NO inhibits cisplatin-induced apoptosis in H-460 cells. transfection, cells were trypsinized, and the cell suspensions were plated Cisplatin is frequently used as a chemotherapeutic agent for solid onto 75-mL culture flasks and cultured for 24 to 28 days with G418 selection tumors and has been reported to induce apoptosis in sensitive A (400 g/mL). The pooled stable transfectant was identified by Western blot cells, including lung epithelial cells (1, 2). To study the role of NO in analysis of Bcl-2 and was cultured in G418-free RPMI for at least two cisplatin-induced apoptosis, we first characterized cell death passages before each experiment. response to cisplatin treatment in human lung epithelial carcino- Apoptosis and cytotoxicity assays. Apoptosis was determined by Hoechst 33342 assay (Molecular Probes) and by DNA fragmentation ELISA ma H-460 cells. Cells were treated with various concentrations of A using a kit from Roche Molecular Biochemicals (Indianapolis, IN). For cisplatin (0-500 mol/L), and apoptosis was determined after 12 Hoechst assay, cells were incubated with 10 Ag/mL Hoechst 33342 for 30 hours by Hoechst 33342 and ELISA-based DNA fragmentation minutes and scoring the percentage of cells having intensely condensed assays. Treatment of the cells with cisplatin caused a dose- chromatin and/or fragmented nuclei by UV microscopy using a Pixera dependent increase in cell apoptosis over control level, as indicated software (Leica Microsystems, Bannockburn, IL). For ELISA assay, cells were by increased nuclear fluorescence and chromatin condensation lysed with 200 AL of lysis buffer, and the cell lysate (20 AL) was mixed with (Fig. 1A). At the treatment dose of 50 Amol/L, f10% of the treated A an antibody solution provided by the supplier (80 L) at room temperature cells showed apoptotic nuclear morphology with the cell death for 2 hours. The substrate was then added after the wells were washed. After response approaching 60% at the treatment dose of 500 Amol/L. incubation for 10 minutes at 37jC, the reaction was stopped, and absor- ELISA results showed a similar dose effect of cisplatin on DNA bance was measured using a microplate reader at a wavelength of 405 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- fragmentation (Fig. 1B). To confirm the death-inducing effect of trazolium bromide (MTT) colorimetric assay using a kit from Roche cisplatin, cells were similarly treated with cisplatin, and cell Molecular Biochemicals (MTT-based Cell Proliferation kit 1). Cells in 96- survival was determined by MTT assay. Consistent with the well plates were incubated with 500 mg/mL of MTT for 4 hours at 37jC. The apoptosis assays, our results showed that cisplatin decreased cell intensity of the MTT product was measured at 550 nm using a microplate survival in a dose-dependent manner (Fig. 1C).

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cisplatin and NAC completely inhibited the apoptotic effect of cisplatin. Likewise, cotreatment of the cells with caspase inhibitor zVAD-fmk completely inhibited the apoptotic effect of cisplatin, indicating the requirement of caspase activation in this process. These results suggest that both ROS and NO play an important role in cisplatin-induced apoptosis through a caspase-dependent mechanism. However, ROS serve as a positive regulator, whereas NO is a negative regulator of cisplatin-induced apoptosis. Modulation of NO and ROS levels in H-460 cells. To provide a relationship between the apoptotic response and NO/ROS modulation by the test agents, we treated the cells with cisplatin and NO/ROS modulators and studied their effects on intracellular NO/ ROS levels by flow cytometry using the NO fluorescent probe DAF- DA and the ROS probe DCF-DA. Figure 2A shows that the NO donor sodium nitroprusside significantly increased the cellular level of NO, whereas the NO inhibitor aminoguanidine inhibited it,

Figure 1. Cisplatin induces cell apoptosis and its inhibition by NO. Subconfluent (90%) monolayers of H-460 cells were treated with cisplatin (0-500 Amol/L) at 37jC for 12 hours. A, dose effect of cisplatin on cell apoptosis determined by Hoechst 33342 assay. B, effect of cisplatin on DNA nucleosomal fragmentation determined by ELISA. C, effect of cisplatin on cell survival determined by MTT assay. D, effect of NO/ROS modulators on cisplatin-induced apoptosis. H-460 cells were pretreated with sodium nitroprusside (SNP; 500 Ag/mL), dipropylenetriamine (DPTA) NONOate (200 Amol/L), aminoguanidine (AG; 300 Amol/L), PTIO (300 Amol/L), NAC (1 mmol/L), or zVAD-fmk (10 Amol/L) for 1 hour. The cells were then either left untreated or treated with cisplatin (200 Amol/L) for 12 hours and analyzed for apoptosis by Hoechst 33342 assay. Columns, mean (n = 4); bars, SD. *, P < 0.05 versus nontreated control; #, P < 0.05 versus cisplatin-treated control.

To investigate the potential role of NO in the regulation of cisplatin-induced apoptosis, cells were treated with various NO inhibitors and donors followed by cisplatin treatment. Figure 1D shows that the NO synthase inhibitor aminoguanidine and the NO scavenger PTIO effectively increased the cellular response to cisplatin-induced cell death, whereas the NO donors sodium nitroprusside and dipropylenetriamine NONOate had an opposite Figure 2. Effects of cisplatin and NO/ROS modulators on cellular NO and ROS effect. The NO-modulating agents, when used alone at the levels. A, subconfluent (90%) monolayers of H-460 cells were pretreated with aminoguanidine (AG; 300 Amol/L) or sodium nitroprusside (SNP; 500 Ag/mL) for indicated concentrations, had no significant effect on cell apoptosis 1 hour, after which they were treated with cisplatin (CP; 200 Amol/L) and (Fig. 1D). These results indicate that NO plays a role as a negative analyzed for NO levels by flow cytometry using the fluorescent probe DAF-DA as described in Materials and Methods. The plots show peak NO response at regulator of cisplatin-induced apoptosis. Because previous studies 1 hours post-cisplatin treatment. Representative results with the corresponding have shown that cisplatin induces cell death via an ROS-dependent region indicated by an arrow. B, cells were pretreated with the ROS scavenger mechanism (9, 30), we also tested whether ROS inhibition by the NAC (1 mmol/L) for 1 hour and then exposed to cisplatin (200 Amol/L) for 1 hour. ROS production was determined by flow cytometry using the fluorescent antioxidant NAC could prevent cisplatin-induced apoptosis in our oxidative probe DCF-DA. Columns, mean (n =4);bars, SD. *, P < 0.05 versus cell system. Figure 1D shows that cotreatment of the cells with nontreated control; #, P < 0.05 versus cisplatin-treated control. www.aacrjournals.org 6355 Cancer Res 2006; 66: (12). June 15, 2006

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Cancer Research as indicated by the corresponding changes in DAF fluorescence intensity. The antioxidant NAC completely inhibited cisplatin- induced ROS generation as shown by the decrease in DCF fluorescence intensity (Fig. 2B). Treatment of the cells with cisplatin alone had an inhibitory effect on cellular NO level but increased the ROS level over nontreated controls (Fig. 2A and B). These results support our earlier finding and indicate the regulatory role of NO and ROS in cisplatin-induced cell death. Bcl-2 expression determines cell death resistance to cisplatin in H-460 cells. Aberrant expression of the antiapoptotic Bcl-2 protein has been shown to mediate cisplatin resistance in many cell types (9–12). To test whether Bcl-2 is involved in apoptosis resistance to cisplatin in H460 cells, we stably transfected the cells with Bcl-2 or control plasmid, and their effect on cisplatin- induced apoptosis was determined. Figure 3A shows that overexpression of Bcl-2 significantly inhibited apoptosis over a wide concentration range of cisplatin treatment compared with mock transfection controls. Western blot analysis of Bcl-2 shows an increased expression of the protein in Bcl-2-transfected cells but not in mock-transfected cells (Fig. 3B). These results indicate the role of Bcl-2 in cell death resistance to cisplatin. NO modulates Bcl-2 expression in H-460 cells. Having shown the role of NO and Bcl-2 in cisplatin-induced apoptosis, we next investigated the potential regulation of Bcl-2 by NO in H-460 cells. Cells were treated with cisplatin in the presence or absence of NO modulators and Bcl-2 expression was determined by immunoblotting after 12 hours. Figure 4A shows that cisplatin treatment caused a significant decrease in Bcl-2 expression, which was further decreased upon the addition of the NO inhibitors aminoguanidine and PTIO. In contrast, treatment of the cells with the NO donor sodium nitroprusside or dipropylenetriamine NONOate completely inhibited cisplatin-induced Bcl-2 down-regulation and further increased the Bcl-2 expression over nontreated control level. The modulating effect

Figure 4. NO inhibits cisplatin-induced Bcl-2 down-regulation. A, subconfluent monolayers of H-460 cells were pretreated for 1 hour with aminoguanidine (AG; 300 Amol/L), PTIO (300 Amol/L), sodium nitroprusside (SNP; 500 Ag/ml), or dipropylenetriamine (DPTA) NONOate (200 Amol/L). The cells were then treated with cisplatin (CP; 200 Amol/L) for 12 hours, and cell extracts were prepared and analyzed for Bcl-2 by immunoblotting. Blots were reprobed with h-actin antibody to confirm equal loading of samples. The immunoblot signals were quantified by densitometry, and mean data from independent experiments were normalized to the result obtained in cells in the absence of cisplatin (control). B, dose effect of NO modulators on Bcl-2 expression. Cells were pretreated with varying doses of aminoguanidine (100, 200, and 300 Amol/L) or sodium nitroprusside (100, 250, and 500 Ag/mL) for 1 hour, after which they were either left untreated or treated with cisplatin (200 Amol/L) for 12 hours. Cell lysates were prepared and analyzed for Bcl-2 expression by immunoblotting. C, cells were pretreated with lactacystin (20 Amol/L), NAC (1 mmol/L), or zVAD-fmk (10 Amol/L) for 1 hour followed by cisplatin treatment (200 Amol/L) for 12 hours. Columns, mean (n = 4); bars, SD. *, P < 0.05 versus nontreated control; #, P < 0.05 versus cisplatin-treated control.

of NO on Bcl-2 expression was dose dependent as shown by the gradual decrease and increase in Bcl-2 levels upon treatment with the increasing doses of the NO inhibitor aminoguanidine and NO donor sodium nitroprusside, respectively (Fig. 4B). Figure 3. Bcl-2 overexpression increases cell death resistance to cisplatin. Because previous studies have shown that Bcl-2 is rapidly down- A, H-460 cells were stably transfected with a myc-tagged Bcl-2 plasmid or a regulated by proteasomal degradation via an ROS-dependent control pcDNA3 plasmid as described in Materials and Methods. Transfected cells were treated with increasing doses of cisplatin (0-500 Amol/L) for 12 hours, pathway (9, 17), we therefore investigated whether Bcl-2 down- and apoptosis was determined by Hoechst 33342 assay. *, P < 0.05 versus regulation by cisplatin is also mediated by this pathway. Cells were mock transfection. B, Western blot analysis of Bcl-2 expression in mock and treated with lactacystin, a highly specific proteasome inhibitor, and Bcl-2 transfected H-460 cells. Cell extracts were prepared and separated on 10% polyacrylamide-SDS gels, transferred, and probed with Bcl-2 and myc its effect on cisplatin-induced Bcl-2 down-regulation was examined antibodies. h-Actin was used as a loading control. by immunoblotting. Figure 4C shows that lactacystin completely

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. NO Increases Cisplatin Resistance inhibited Bcl-2 down-regulation by cisplatin, indicating the dominant role of proteasomal degradation in the down-regulation process. The result was confirmed by the observation that another proteasome inhibitor (MG132) also inhibited the decrease in Bcl-2 expression caused by cisplatin (data not shown). Figure 4B also shows that the antioxidant NAC was able to inhibit Bcl-2 down- regulation by cisplatin, supporting the role of ROS in the degradation process. Bcl-2 has also been reported to be enzymatically cleaved by caspase-3 in some cells (31). To test whether this process might be involved in cisplatin-induced Bcl-2 degradation in H-460 cells, we treated the cells with caspase inhibitor zVAD-fmk and studied its effect on Bcl-2 expression by immunoblotting. Figure 4C shows that the caspase inhibitor was unable to inhibit Bcl-2 down-regulation induced by cisplatin, indicating the insignificant role of this enzyme in cisplatin-induced Bcl-2 degradation. NO does not affect Bcl-2 phosphorylation in H-460 cells. Bcl- 2 stability is mainly regulated by phosphorylation with the Ser87 phosphorylation playing a dominant role in this process (16, 17). Apoptotic stimuli, such as TNF-a and ROS, induce dephosphorylation of Ser87, which promotes proteasomal degradation of Bcl-2 (9, 16, 17). To investigate the potential mechanism of NO regulation of Bcl-2, we tested the effect of NO modulators on Bcl-2 phosphorylation in cisplatin-treated cells. As control, cells were treated with the ROS scavenger NAC, and its effect on Bcl-2 phosphorylation was determined by Western blotting. The results show that cisplatin strongly induced Bcl-2 dephosphorylation at Ser87, and NAC completely inhibited this dephosphorylation (Fig. 5A). In contrast, neither of the NO donors (sodium nitroprusside and dipropylenetriamine NONOate) nor NO inhibitors (aminoguanidine and PTIO) was able to significantly affect this dephosphorylation (Fig. 5A). These results suggest that NO regulates Bcl-2 stability via a different mechanism from ROS, and that this regulation is phosphorylation-independent. NO prevents cisplatin-induced ubiquitination of Bcl-2. To further investigate the mechanism by which NO protects cisplatin- induced Bcl-2 degradation, we analyzed ubiquitination of Bcl-2 in Figure 5. Effects of NO modulators on Bcl-2 phosphorylation and ubiquitination. response to cisplatin treatment by immunoprecipitation. Cells A, subconfluent monolayers of H-460 cells were pretreated for 1 hour with sodium nitroprusside (SNP; 500 Ag/mL), dipropylenetriamine (DPTA) NONOate were cotransfected with myc-tagged Bcl-2 and ubiquitin expression (200 Amol/L), aminoguanidine (AG; 300 Amol/L), PTIO (300 Amol/L), or NAC plasmids, and 36 hours later, they were treated with cisplatin in the (1 mmol/L). The cells were then treated with cisplatin (CP; 200 Amol/L) for 12 hours, and cell lysates were prepared and analyzed for Bcl-2 phosphorylation presence or absence of NO donors or inhibitors. After the using phospho-specific Bcl-2 (Ser87) antibody. Densitometry was done to treatments, cell lysates were prepared and immunoprecipitated determine the relative levels of Bcl-2 phosphorylation after reprobing with h-actin using an anti-myc antibody. The resulting immune complexes were antibody. B, H-460 cells were transiently transfected with ubiquitin and myc-tagged Bcl-2 plasmids. Thirty-six hours later, the cells were pretreated with then analyzed for ubiquitin by Western blot using an anti-ubiquitin the same concentrations of NO modulators for 1 hour followed by cisplatin antibody. The results show that cisplatin was able to induce treatment in the presence of lactacystin (10 Amol/L). Cell lysates were ubiquitination of Bcl-2 in a dose-dependent manner, and that the immunoprecipitated with anti-myc antibody, and the immune complexes were analyzed for ubiquitin by Western blotting. Analysis of ubiquitin was performed at NO inhibitors aminoguanidine and PTIO increased this ubiquiti- 2 hours post-cisplatin treatment, where ubiquitination was found to be maximal. nation (Fig. 5B). In contrast, the NO donors sodium nitroprusside Columns, mean (n = 4); bars, SD. *, P < 0.05 versus nontreated control; P and dipropylenetriamine NONOate completely inhibited ubiquiti- #, < 0.05 versus cisplatin-treated control. nation of Bcl-2 (Fig. 5C). These results along with our earlier phosphorylation results indicate that NO inhibited ubiquitination were immunoprecipitated and analyzed by Western blot using an of Bcl-2 through a phosphorylation-independent process. anti-S-nitrosocysteine antibody. Figure 6A shows that treatment of NO induces S-nitrosylation of Bcl-2 and inhibits its the cells with cisplatin resulted in a substantial reduction in ubiquitination. Increasing evidence indicates that NO plays an S-nitrosylated Bcl-2 levels, and cotreatment of the cells with the NO important role in apoptosis through S-nitrosylation of several key donor sodium nitroprusside or dipropylenetriamine NONOate proteins that regulate the apoptosis pathways (32, 33). To determine reversed this effect. Because S-nitrosylation by NO has been whether NO could nitrosylate Bcl-2, which has not been shown, and reported to be inhibited by strong reducing agents, such as DTT whether this process could affect Bcl-2 stability, we did immuno- (34, 35), we further tested whether DTT could prevent the effect of precipitation experiments evaluating the effect NO on Bcl-2 NO on Bcl-2 S-nitrosylation and ubiquitination. The results show S-nitrosylation. Cells expressing ectopic myc-Bcl-2 were treated that DTT was able to prevent S-nitrosylation of Bcl-2 (Fig. 6A) with cisplatin and NO modulators as described, and cell lysates and the ubiquitination effect of NO donors in cisplatin-treated cells www.aacrjournals.org 6357 Cancer Res 2006; 66: (12). June 15, 2006

in many types of tumor cells (9–12). Although the importance of gene expression in regulating apoptotic signal transduction has been emphasized in numerous studies, posttranslational modifications, such as ubiquitination and phosphorylation, have emerged as important regulators of Bcl-2 function (9, 16, 17, 36). However, the mechanisms underlying this regulation and in particular those relevant to cisplatin resistance have not been clearly elucidated. Our results show that treatment of human lung carcinoma H-460 cells with cisplatin resulted in a down-regulation of Bcl-2 and concomitant increase in apoptotic cell death (Figs. 1A and 4A). Down-regulation of Bcl-2 was associated with an increase in ubiquitination and proteasomal degradation of the protein (Figs. 4B and 5B). Overexpression of Bcl-2 protected the cells from cisplatin-induced cell death (Fig. 3), supporting the role of Bcl-2 in cisplatin resistance. Multiple mechanisms of cisplatin resistance in tumor cells have been proposed, including impaired cellular uptake and increased cellular efflux of cisplatin (37), increased DNA lesion repair (38, 39), and defects in mismatch repair that fail to trigger cell death (40–42). Intracellular cisplatin inactivation by redox reactions has also been proposed as a mechanism of cisplatin resistance (43, 44). Increased ROS generation by glutathione depletion or by superoxide dismutase antisense transfection has been shown to prevent Bcl-2-mediated cisplatin resistance (9, 44). Conversely, inhibition of ROS by antioxidants has been shown to increase cisplatin resistance (45). Glutathione can also detoxify cisplatin through the formation of glutathione adducts (43). Cellular glutathione levels can be augmented by NAC, which is converted intracellularly to the rate-limiting substrate for glutathione synthesis. Increased cisplatin resistance by NAC may involve glutathione-cisplatin adduct formation and a corresponding decrease in cisplatin-mediated DNA damage. Alternatively, glutathione may interfere with the apoptotic process through its ability to act as a redox buffer. Indeed, increased glutathione levels have been shown to increase apoptosis resistance to cisplatin through Figure 6. NO induces S-nitrosylation of Bcl-2 and inhibits its ubiquitination by an ROS scavenging mechanism that is independent of cisplatin- cisplatin. A, effect of NO modulators on cisplatin-induced S-nitrosylation of Bcl-2. H-460 cells were transiently transfected with myc-tagged Bcl-2 plasmid. DNA adduct formation or repair (44). Thirty-six hours later, the cells were pretreated with sodium nitroprusside Increasing evidence has also shown that NO plays a role in (SNP; 500 Ag/mL), dipropylenetriamine (DPTA) NONOate (200 Amol/L), apoptosis regulation through its ability to modulate ROS; that is, NO or aminoguanidine (AG; 300 Amol/L) in the presence or absence of DTT (10 mmol/L) for 1 hour. The cells were then treated with cisplatin (CP; 200 Amol/L) can interact with superoxide anion to form peroxynitrite (46, 47) and for 2 hours, and cell lysates were prepared for immunoprecipitation using anti-myc to modify key apoptosis-regulatory proteins through S-nitrosylation antibody. The resulting immune complexes were analyzed for S-nitrosocysteine (32, 33). However, whether or not NO can nitrosylate Bcl-2 and by Western blotting. Densitometry was done to determine the relative S-nitrosocysteine levels after reprobing of the membranes with Bcl-2 antibody. whether it plays a role in cisplatin resistance have not been shown. In B, effect of NO modulators on cisplatin-induced Bcl-2 ubiquitination. Cells were this study, we found that NO can nitrosylate Bcl-2 and prevent its cotransfected with ubiquitin and myc-Bcl-2 plasmids. Thirty-six hours later, they were treated with the indicated test agents in the presence of lactacystin degradation through the ubiquitin-proteasomal pathway. Addition (10 Amol/L). Cell lysates were then immunoprecipitated with anti-myc antibody, of the NO donors sodium nitroprusside and dipropylenetriamine and the immune complexes were analyzed for ubiquitin. Columns, mean (n = 4); NONOate increased Bcl-2 S-nitrosylation (Fig. 6A), decreased its bars, SD. *, P < 0.05 versus cisplatin-treated control; #, P < 0.05 versus NO-modulated controls. ubiquitination and proteasomal degradation (Figs. 4 and 5), and increased cell death resistance to cisplatin (Fig. 1B). In contrast, the NO inhibitors aminoguanidine and PTIO showed opposite effects, (Fig. 6B). These results indicate that S-nitrosylation might be a key thus confirming the role of NO in Bcl-2-mediated cisplatin mechanism used by NO to regulate ubiquitination and proteasomal resistance. It should be noted that although our results indicate degradation of Bcl-2. the nitrosylation of Bcl-2 by NO, such nitrosylation may not be the only essential NO-mediated modification of apoptosis regulatory proteins that suppresses cisplatin-induced cell death. Discussion The mechanism by which S-nitrosylation prevents ubiquitination The antiapoptotic function of Bcl-2 is tightly associated with its of Bcl-2 is unclear but may involve conformational change of S- expression levels, and amplification of this protein has been linked nitrosylated Bcl-2 protein, which prevents its recognition and to resistance to cell death induced by various DNA-damaging subsequent attachment of ubiquitin by the enzyme ubiquitin ligases. agents, including those used in cancer chemotherapy. Aberrant Conformational changes of Bcl-2 by phosphorylation have been expression of Bcl-2 has been shown to mediate cisplatin resistance reported to affect its ubiquitination and stability (48). Moreover,

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. NO Increases Cisplatin Resistance dephosphorylation of Bcl-2 by ROS and TNF-a has been shown to be the early apoptosis process, caspase activity was still low and required for the ubiquitination and degradation of Bcl-2 (9, 16, 17). insufficient to affect Bcl-2 stability, whereas in late apoptosis, Our results on the inhibitory effects of the ROS scavenger NAC on caspase activity was elevated and was able to induce Bcl-2 cleavage. Bcl-2 dephosphorylation and degradation (Figs. 4 and 5) support this Our results on the ubiquitination and proteasomal degradation of finding and further indicate the involvement of ROS in cisplatin- Bcl-2 and the lack of caspase-mediated cleavage at the early time induced Bcl-2 instability. Unlike ROS, however, we found that NO period (>12 hours) support the previous finding and indicate that had no effect on Bcl-2 phosphorylation (Fig. 5), whereas it exhibited proteasomal degradation is a key early event in controlling Bcl-2 strong effects on ubiquitination and degradation (Figs. 4 and 5). stability. These results indicate that dephosphorylation of Bcl-2 might not be In summary, our data provide evidence that NO plays an a necessary event for the triggering of ubiquitination, and that NO important role in regulating cell death resistance to cisplatin. and ROS may regulate Bcl-2 stability via different mechanisms. Cisplatin induces down-regulation of Bcl-2 through proteasome- Alternatively, ROS, through its ability to interact with NO, may mediated degradation. NO negatively regulates this process decrease the availability and thus nitrosylation activity of NO, which through its ability to nitrosylate the protein and inhibit its results in increased Bcl-2 ubiquitination. The ability of the reducing ubiquitination. In showing the S-nitrosylation of Bcl-2, we agent DTT to inhibit both S-nitrosylation and ubiquitination of Bcl-2 document a novel layer of regulation that links NO signaling with (Fig. 5) further supports the role of S-nitrosylation and its regulation Bcl-2-mediated chemoresistance, which represents an important by ROS in the ubiquitination process. mechanism in the control of tumor development and progression. Bcl-2 has been shown to be enzymatically cleaved by caspase-3 Because increased NO production and Bcl-2 expression have been in some cells (31), and NO could inhibit this process by associated with several human tumors, NO may be one of the key S-nitrosylation of the enzyme (49). However, our caspase inhibition regulators of cell death resistance and tumor growth through S- study failed to detect the inhibitory effect of the caspase inhibitor nitrosylation. This finding on the novel function of NO in Bcl-2 zVAD-fmk on Bcl-2 expression (Fig. 4B), although this inhibitor regulation may have important implications in cancer chemother- completely inhibited cisplatin-induced apoptosis (Fig. 1D). The apy and prevention. likely explanation for the observed discrepancy may be the difference in experimental conditions and cell type used. In Jurkat cells and prostate cancer PC-3 cells, caspase-3 inhibitor was also Acknowledgments unable to prevent Bcl-2 cleavage, whereas proteasome inhibitors Received 12/19/2005; revised 4/12/2006; accepted 4/19/2006. were found to prevent Bcl-2 degradation (36). In H-460 cells, Grant support: NIH grants HL-071545 and HL076340 (Y. Rojanasakul) and caspase-mediated Bcl-2 cleavage was observed but only after 12 Thailand Research Fund RGJ grant 5.Q.CU.46/A.1 (U. Nimmannit). The costs of publication of this article were defrayed in part by the payment of page hours of drug treatment, whereas proteasome-mediated cleavage charges. This article must therefore be hereby marked advertisement in accordance was also observed at earlier times (48). It was suggested that during with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Nitric Oxide Regulates Cell Sensitivity to Cisplatin-Induced Apoptosis through S-Nitrosylation and Inhibition of Bcl-2 Ubiquitination

Pithi Chanvorachote, Ubonthip Nimmannit, Christian Stehlik, et al.

Cancer Res 2006;66:6353-6360.

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